PURPOSE: To determine the feasibility of confocal profiling in measuring surface roughness and obtaining 3-dimensional reconstructions of mechanically dissected and femtosecond (fs)-laser photodisrupted endothelial lamellae. To determine the predictability of single-pass dissection of ultrathin endothelial lamellae using a novel motor-driven linear microkeratome. METHODS: Thirty (n = 30) human corneas were harvested using a motor-driven linear microkeratome (n = 20); a hand-driven rotatory microkeratome (n = 6); and a 60-kHz fs laser (n = 4). Surface roughness was measured using an optical profiler operated in confocal microscopy mode followed by environmental scanning-electron-microscopy. RESULTS: Mean surface roughness for the fs laser, motor-driven linear microkeratome, and hand-driven rotatory microkeratome measured 1.90 ± 0.48 µm, 1.06 ± 0.42 µm, and 0.93 ± 0.25 µm, respectively. Femtosecond photodisrupted lamellae were significantly rougher than mechanically dissected lamellae (P <0.001). Mean (±SD) cutting depth with the motor-driven linear microkeratome measured: 552 ± 11 µm (550-µm head); 505 ± 19 µm (550-µm head); 459 ± 19 µm (450-µm head); and 392 ± 20 µm (400-µm head). CONCLUSIONS: Confocal microscopy allows quantitative surface roughness analysis and 3-dimensional reconstruction of human corneal lamellae. Femtosecond-laser photodisruption at 60 kHz results in rougher surfaces compared with mechanical dissection. The motor-driven linear microkeratome allows single-pass dissection of ultrathin endothelial lamellae with a standard deviation =20 µm.